Method of preparing cow brain homogenate

ABSTRACT

An assay method is disclosed which isolates and detects the presence of a disease related conformation of a protein (e.g., PrP Sc ) present in a sample also containing the non-disease related conformation of the protein (e.g., PrP Sc ). The sample is treated (e.g., contacted with protease) in a manner which hydrolyzes the disease related conformation and not the non-disease related conformation. The treated sample is contacted with a binding partner (e.g., a labeled antibody which binds PrP Sc ) and the occurrence of binding provides and indication that PrP Sc  is present. Alternatively the PrP Sc  of the treated sample is denatured (e.g., contacted with guanadine) or unfolded. The unfolded PrP Sc  is contacted with a binding partner and the occurrence of binding indicates the presence of PrP Sc  in the sample. In another embodiment, PrP Sc  and PrP C  are reacted with a labeled antibody that binds both conformations and a conformation that binds only the disease related conformation, and the presence of the disease related conformation is determined by comparing the two.

CROSS-REFERENCES

This application is a continuation of U.S. application Ser. No.11/054,585 filed Feb. 8, 2005 which is a continuation of U.S.application Ser. No. 10/742,241 filed Dec. 18, 2003, now issued U.S.Pat. No. 6,875,577 on Apr. 5, 2005 which is a continuation of U.S.application Ser. No. 10/047,431, filed Jan. 14, 2002, now issued U.S.Pat. No. 6,677,125 on Jan. 13, 2004 which is a continuation applicationof 09/754,443, filed Jan. 3, 2001, now issued U.S. Pat. No. 6,406,864 onJun. 18, 2004 which is a continuation application of U.S. applicationSer. No. 09/169,574, filed Oct. 9, 1998, now issued U.S. Pat. No.6,214,565 and further is a continuation-in-part of U.S. application Ser.No. 09/026,967, filed Feb. 20, 1998, issued as U.S. Pat. No. 5,977,324on Nov. 2, 1999, all of which are incorporated herein by reference intheir entirety and to which applications is claimed priority under 35U.S.C. §120.

GOVERNMENT RIGHTS

The United States Government may have certain rights in this applicationpursuant to Grant No. AG02132, AG10770, NS22786, NS14069, and NS07219awarded by the National Institutes of Health.

FIELD OF THE INVENTION

This invention relates to the field of bioassays and more particularlyto an assay which makes it possible to isolate and detect a diseaseconformation of a protein present in a native sample also containing anon-disease conformation of the protein.

BACKGROUND OF THE INVENTION

Prions are infectious pathogens that cause invariably fatal priondiseases (spongiform encephalopathies) of the central nervous system inhumans and animals. Prions differ significantly from bacteria, virusesand viroids. The dominating hypothesis is that no nucleic acid isnecessary to allow for the infectivity of a prion protein to proceed.

A major step in the study of prions and the diseases they cause was thediscovery and purification of a protein designated prion protein[Bolton, McKinley et al. (1982) Science 218:1309-1311; Prusiner, Boltonet al. (1982) Biochemistry 21:6942-6950; McKinley, Bolton et al. (1983)Cell 35:57-62]. Complete prion protein-encoding genes have since beencloned, sequenced and expressed in transgenic animals. PrP^(C) isencoded by a single-copy host gene [Basler, Oesch et al. (1986) Cell46:417-428] and when PrP^(C) is expressed it is generally found on theouter surface of neurons. Many lines of evidence indicate that priondiseases results from the transformation of the normal form of prionprotein (PrP^(C)) into the abnormal form (PrP^(Sc)). There is nodetectable difference in the amino acid sequence of the two forms.However, PrP^(Sc) when compared with PrP^(C) has a conformation withhigher β-sheet and lower α-helix content [Pan, Baldwin et al. (1993)Proc Natl Acad Sci USA 90:10962-10966; Safar, Roller et al. (1993) JBiol Chem 268:20276-20284]. The presence of the abnormal PrP^(Sc) formin the brains of infected humans or animals is the only disease-specificdiagnostic marker of prion diseases.

PrP^(Sc) plays a key role in both transmission and pathogenesis of priondiseases (spongiform encephalopathies) and it is a critical factor inneuronal degeneration [Prusiner (1997) The Molecular and Genetic Basisof Neurological Disease, 2nd Edition: 103-143]. The most common priondiseases in animals are scrapie of sheep and goats and bovine spongiformencephalopathy (BSE) of cattle [Wilesmith and Wells (1991) Curr TopMicrobiol Immunol 172:21-38]. Four prion diseases of humans have beenidentified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3)Gerstmann-Streussler-Sheinker Disease (GSS), and (4) fatal familialinsomnia (FFI) [Gajdusek (1977) Science 197:943-960; Medori, Tritschleret al. (1992) N Engl J Med 326:444-449]. Initially, the presentation ofthe inherited human prion diseases posed a conundrum which has sincebeen explained by the cellular genetic origin of PrP.

Prions exist in multiple isolates (strains) with distinct biologicalcharacteristics when these different strains infect in geneticallyidentical hosts [Prusiner (1997) The Molecular and Genetic Basis ofNeurological Disease, 2nd Edition: 165-186]. The strains differ byincubation time, by topology of accumulation of PrP^(Sc) protein, and insome cases also by distribution and characteristics of brain pathology[DeArmond and Prusiner (1997) Greenfield's Neuropathology, 6thEdition:235-280]. Because PrP^(Sc) is the major, and very probably theonly component of prions, the existence of prion strains has posed aconundrum as to how biological information can be enciphered in amolecule other than one comprised of nucleic acids. The partialproteolytic treatment of brain homogenates containing some prionisolates has been found to generate peptides with slightly differentelectrophoretic mobilities [Bessen and Marsh (1992) J Virol66:2096-2101; Bessen and Marsh (1992) J Gen Virol 73:329-334; Telling,Parchi et al. (1996) Science 274:2079-2082]. These findings suggesteddifferent proteolytic cleavage sites due to the different conformationof PrP^(Sc) molecules in different strains of prions. Alternatively, theobserved differences could be explained by formation of differentcomplexes with other molecules, forming distinct cleavage sites inPrP^(Sc) in different strains [Marsh and Bessen (1994) Phil Trans R SocLond B 343:413-414]. Some researchers have proposed that different prionisolates may differ in the glycosylation patterns of prion protein[Collinge, Sidle et al. (1996) Nature 383:685-690; Hill, Zeidler et al.(1997) Lancet 349:99-100]. However, the reliability of bothglycosylation and peptide mapping patterns in diagnostics of multipleprion strains is currently still debated [Collings, Hill et al. (1997)Nature 386:564; Somerville, Chong et al. (1997) Nature 386:564].

A system for detecting PrP^(Sc) by enhancing immunoreactivity afterdenaturation is provided in Serban, et al., Neurology, Vol. 40, No. 1,Ja 1990. Sufficiently sensitive and specific direct assay for infectiousPrP^(Sc) in biological samples could potentially abolish the need foranimal inoculations completely. Unfortunately, such does not appear tobe possible with current PrP^(Sc) assays—it is estimated that thecurrent sensitivity limit of proteinase-K and Western blot-basedPrP^(Sc) detection is in a range of 1 μg/ml which corresponds to 10⁴-10⁵prion infectious units. Additionally, the specificity of the traditionalproteinase-K-based assays for PrP^(Sc) is in question in light of recentfindings of only relative or no proteinase-K resistance of undoubtedlyinfectious prion preparations [Hsiao, Groth et al. (1994) Proc Natl AcadSci USA 91:9126-9130] Telling, et al. (1996) Genes & Dev.

Human transthyretin (TTR) is a normal plasma protein composed of fouridentical, predominantly β-sheet structured units, and serves as atransporter of hormone thyroxine. Abnormal self assembly of TTR intoamyloid fibrils causes two forms of human diseases, namely senilesystemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP)[Kelly (1996) Curr Opin Strut Biol 6(1):11-7]. The cause of amyloidformation in FAP are point mutations in the TTR gene; the cause of SSAis unknown. The clinical diagnosis is established histologically bydetecting deposits of amyloid in situ in biopsy material.

To date, little is known about the mechanism of TTR conversion intoamyloid in vivo. However, several laboratories have demonstrated thatamyloid conversion may be simulated in vitro by partial denaturation ofnormal human TTR [McCutchen, Colon et al. (1993) Biochemistry 32(45):12119-27; McCutchen and Kelly (1993) Biochem Biophys Res Commun 197(2)415-21]. The mechanism of conformational transition involves monomericconformational intermediate which polymerizes into linear β-sheetstructured amyloid fibrils [Lai, Colon et al. (1996) Biochemistry35(20):6470-82]. The process can be mitigated by binding withstabilizing molecules such as thyroxine or triiodophenol [Miroy, Lai etal. (1996) Proc Natl Acad Sci USA 93(26):15051-6].

In view of the above points, there is clearly a need for a specific,high flow-through, and cost-effective assay for testing sample materialsfor the presence of a pathogenic protein including transthyretin andprion protein.

SUMMARY OF THE INVENTION

The assay of the invention involves treating a sample suspected ofcontaining a protein in at least two conformations, i.e., in both adisease conformation and a non-disease conformation. The sample istreated with a compound which hydrolyzes the non-disease relatedconformation of the protein but neither hydrolyzes or denatures thedisease conformation of the protein. After treatment the assay canproceed in two possible ways. In a first method the sample is broughtinto contact with a binding agent such as an antibody which binds to thedisease conformation of the protein so that any detected bindingindicates the presence of protein in the disease conformation beingpresent in the sample. In a second method the treated sample is thensubjected to a second treatment step which at least partially denaturesthe disease conformation of the protein so that the denatured proteinwill bind to a wider range of binding partners. After denaturation thesample is brought into contact with a binding partner which binds thedenatured, diseased conformation of the protein.

Depending on the steps used in the assay of the invention one of twotypes of antibodies may be used. Accordingly, both basic types of assaysthe sample is treated with a compound, e.g. a metalloendopeptidase,which selectively hydrolyzes PrP^(C) but not PrP^(Sc). Thereafter, thetreated sample can be subjected to two different types of processing,each of which uses a generally different type of antibody.

The first general type of antibody selectively binds to the diseaseconformation of the protein. For example, antibodies that selectivelyrecognize PrP^(Sc) bind to an epitope on the C-terminus of the protein.When a PrP protein is in its PrP^(Sc) configuration its C-terminus canbe bound by antibodies of the type described in WO 97/10505 publishedMar. 20, 1997—reference is also made to WO 98/37210 which claims todisclose antibodies which bind PrP^(Sc). Both of these PCT publicationsare incorporated herein by reference to describe and disclose antibodiesand method of making antibodies.

The second general type of antibody binds to both the disease and thenon-disease conformations of the protein. For example, antibodies thatrecognize an epitope on the N-terminus of the PrP protein recognize bothPrP^(Sc) and PrP^(C) following denaturation of the proteins. When thePrP protein is in the PrP^(Sc) configuration the N-terminus is notexposed and as such can not be bound by an antibody. To expose anepitope of the N-terminus the PrP^(Sc) is denatured, e.g. by exposure toguanadine HCl under conditions (pH, temperature, and time) which causesthe PrP^(Sc) to unfold or change its 3-dimensional structure such that aC-terminal epitope is exposed. In this unfolded configuration a widerange of binding partner including commercially available antibodies canbe used for detection. Since such antibodies also bind PrP^(C) all ofthe PrP^(C) must be removed, e.g., by selective hydrolysis.

An example of an antibody which binds an epitope of the N-terminus isthe monoclonal antibody 3F4 produced by the hybridoma cell line ATCCHB9222 deposited on Oct. 8, 1986 in the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852 and disclosed anddescribed in U.S. Pat. No. 4,806,627 issued Feb. 21, 1989—incorporatedby reference to disclose antibodies which selectively bind PrP^(C). Inaddition to antibody other binding partners which bind the non-diseaserelated conformation but not the disease related conformation could beused in the assay of the invention. Antibodies such as 3F4 and othersused in the assays described in the examples are commercially available.

In one embodiment of the invention, one portion of a sample containingtwo conformations of a protein (e.g. PrP^(C) and PrP^(Sc)) is reactedwith a binding partner (e.g. R1) that binds both conformations, andanother portion of the same sample is reacted with a binding partner(e.g. 3F4) that binds only one of the two forms (e.g. PrP^(C)). Thedisease related conformation is determined by comparing the two. If thebinding partner which binds both conformations shows more binding thanthe binding partner which binds only one conformation, this shows thatboth conformations are present in the sample. For example, if R1 bindsto more protein than 3F4, PrP^(Sc) is present in the sample. Nohydrolysis treatment is needed with this method. However, pretreatmentmay be used and comparison of the binding may be adjusted for a varietyof factors, e.g. binding affinities, comparisons to known samples,hybridization times, variations in signal due to secondary antibodies,etc.

An aspect of the invention is to provide an immunoassay which isapplicable to assaying samples containing proteins, which samples aresuspected of containing a protein which occurs within a nativenon-disease conformation and a disease related conformation (e.g., PrPprotein, βA4 protein and transthyretin).

Another aspect of the invention is to provide an assay whichdifferentiates between (1) disease related proteins or portions thereofwhich are not hydrolyzed by limited protease treatment with a proteasesuch as proteinase K (protease resistant proteins, e.g. PrP 27-30) and(2) disease related proteins which are hydrolyzed by a limited proteasetreatment with a protease such as proteinase K (e.g., protease-sensitivePrP^(Sc)).

An advantage of the present invention is that the immunoassay canquickly and accurately determine the presence of proteins in the diseaserelated conformation (e.g., PrP^(Sc), βA4 and transthyretin) even thoughthe antibody used in the assay does not bind or has a very low degree ofbinding affinity for the protein in the disease related conformation andthe disease related conformation is present in a lower concentrationthan the non-disease conformation.

A feature of the invention is that the signal obtained can be enhancedby the use of transgenic animals, e.g., mice which are used to detectthe presence of a protein in a sample.

Another feature is that time-resolved, dissociation-enhancedfluorescence or a dual wavelength, laser driven fluorometer can be usedto enhance sensitivity.

Another advantage is that the assay can detect levels of the diseasecausing conformation of a protein at a concentration of 1×10³particles/ml or less.

A specific object is to provide a diagnostic assay for determining thepresence of infectious prion protein in variable sample materialsobtained or derived from human, primate, monkey, pig, bovine, sheep,goat, deer, elk, cat, dog, mouse, chicken, and turkey tissues and/orbody fluids.

Another specific object is to provide a diagnostic assay for determiningthe presence of βA4 protein in variable sample materials obtained orderived from human, primate, monkey, pig, bovine, sheep, goat, deer,elk, cat, dog, mouse, chicken, and turkey tissues and/or body fluids.

Another object is to provide a rapid assay for native infectious prionprotein in the brains of transgenic and non-transgenic animals injectedwith sample material potentially containing prions.

Another object is to provide a method to evaluate decontaminationprocedures by assaying the level of denaturation of pathogenic proteins(e.g., prions or β-sheet βA4) after such treatments.

Another advantage is that the process can be carried out without anantibody directly able to recognize an infectious conformation of aprotein, and without using a proteinase K step to eliminate the signalof normal (non-disease) isoforms of the protein such as PrP^(C).

Another advantage is that in the invented process there is no need forthe antibody directly able to recognize a pathogenic conformation of βA4or transthyretin.

An important feature of the assay is the rapid, cost effective and highflow-through design which can be designed with the capacity to screen 96samples per day per 96 well plate.

Another aspect of the invention is the diagnostic method toquantitatively detect TTR in the abnormal, amyloid conformation insample material obtained from human and animal tissues, body fluids, andpharmaceuticals.

The invented process provides a direct, sensitive method to distinguishand quantify the normal and amyloid conformations of TTR in a mixturepresent in sample materials.

An important object is to provide specific diagnostic assay forpathogenic TTR in variable sample materials obtained or derived fromhuman, primate, monkey, pig, bovine, sheep, deer, elk, cat, dog, andchicken tissues.

Another object is to provide a rapid assay for amyloid form of TTR intransgenic animals.

The specific advantage is that invented assay may detect pathogenicforms of TTR in a mixture with denatured nonpathogenic forms of the sameor in a mixture with a soluble form of TTR—for example, detect less than1×10³ particles per ml.

These and other objects, advantages, and features of the inventedprocess will become apparent to those skilled in the art upon readingthe details of the assay method, antibody development and testing, andtransgenic mouse as more fully described below with reference to theattached figures.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before the present assays and methods are disclosed and described, it isto be understood that this invention is not limited to particularantibodies, proteins, labels, assays or methods as such may, of coursevary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

The publications discussed herein are provided solely for the disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the present invention is notentitled to antedate such publication by virtue of prior invention.Further, the dates of the publications provided are subject to change ifit is found that the actual date of publication is different from thatprovided here.

Definitions

The term “protein” as used herein is intended to encompass any aminoacid sequence and include modified sequences such as glycoproteins. Theterm includes naturally occurring proteins and peptides as well as thosewhich are recombinantly or synthetically synthesized. As used inconnection with the present invention the term “protein” is specificallyintended to cover naturally occurring proteins which occur in at leasttwo different conformations wherein both conformations have the same orsubstantially the same amino acid sequence but have different threedimensional structures. The two conformations of the protein include atleast one conformation which is not related to a disease state and atleast one conformation which is related to a disease state—pathogenic. Aspecific and preferred example of a protein as used in connection withthis disclosure is a PrP protein which includes the non-disease formreferred to as the PrP^(c) form and the disease related form referred asthe PrP^(Sc). Although a prion protein or the PrP^(Sc) form of a PrPprotein is infectious and pathogenic, the disease conformation of otherproteins is not infectious although it is pathogenic. As used herein,the term pathogenic may mean that the protein actually causes thedisease or it may simply mean that the protein is associated with thedisease and therefore is present when the disease is present. Thus, apathogenic protein as used in connection with this disclosure is notnecessarily a protein which is the specific causative agent of adisease.

The terms “pretreatment”, “unfolding treatment”, and “limited proteasetreatment” are intended to encompass the descriptions and use of theseterms as provided in the respective sections having these headings.

The terms “PrP protein”, “PrP” and like are used interchangeably hereinand shall mean both the infectious particle form PrP^(Sc) known to causediseases (spongiform encephalopathies) in humans and animals and thenoninfectious form PrP^(C) which, under appropriate conditions isconverted to the infectious PrP^(Sc) form.

The terms “prion”, “prion protein” and “PrP^(Sc) protein” and the likewe used interchangeably herein to refer to the infectious PrP^(Sc) formof PrP, and is a contraction of the words “protein” and “infection.”Particles are comprised largely, if not exclusively, of PrP^(Sc)molecules encoded by a PrP gene. Prions are distinct from bacteria,viruses and viroids. Known prions infect animals to cause scrapie, atransmissible, degenerative disease of the nervous system of sheep andgoats, as well as bovine spongiform encephalopathy (BSE), or “mad cowdisease”, and feline spongiform encephalopathy of cats. Four priondiseases known to affect humans are (1) kuru, (2) Creutzfeldt-JakobDisease (CJD), (3) Gerstmann-Straussler-Scheinker Disease (GSS), and (4)fatal familial insomnia (FFI). As used herein “prion” includes all formsof prions causing all or any of these diseases or others in any animalsused—and in particular in humans and domesticated farm animals.

The term “PrP gene” is used herein to describe genetic material whichexpresses proteins including known polymorphisms and pathogenicmutations. The term “PrP gene” refers generally to any gene of anyspecies which encodes any form of a PrP protein. Some commonly known PrPsequences are described in Gabriel et al., Proc. Natl. Acad. Sci. USA89:9097-9101 (1992), and U.S. Pat. Nos. 5,565,186; 5,763,740; 5,792,901;and WO97/04814, incorporated herein by reference to disclose anddescribe such sequences. The PrP gene can be from any animal, includingthe “host” and “test” animals described herein and any and allpolymorphisms and mutations thereof, it being recognized that the termsinclude other such PrP genes that are yet to be discovered. The proteinexpressed by such a gene can assume either a PrP^(C) (non-disease) orPrP^(Sc) (disease) form.

The term “binding partner” refers to any molecule which binds the targetmolecule of interest. Preferably, the binding is of sufficiently highaffinity as to make it possible to bind target molecules of interestpresent in a low concentration, e.g., 1×10³, particles per ml or less.More preferably the binding partner is selective in binding only thetarget molecule and not other molecules. Preferred binding partners areantibodies as defined below.

The term “antibody” stands for an immunoglobulin protein which iscapable of binding an antigen. Antibody as used herein is meant toinclude the entire antibody as well as any antibody fragments (e.g.F(ab)′, Fab, Fv) capable of binding the epitope, antigen or antigenicfragment of interest. Antibodies for assays of the invention may beimmunoreactive or immunospecific for and therefore specifically andselectively bind to a protein of interest e.g., an A4β amyloid proteinor a PrP protein. Antibodies which are immunoreactive and immunospecificfor both the native non-disease form and the treated disease form butnot for the untreated disease form, (e.g., for both native PrP^(C) andtreated PrP^(Sc) but not native PrP^(Sc)) may be used because the sampleis treated to remove i.e., hydrolyze PrP^(C). Antibodies for PrP arepreferably immunospecific—e.g., not substantially cross-reactive withrelated materials. Some specific antibodies which can be used inconnection with the invention are disclosed in published PCT applicationWO 97/10505 which is incorporated herein by reference to disclose anddescribe antibodies. This published PCT application corresponds to U.S.Ser. No. 08/713,939. Antibodies disclosed in the PCT application whichbind PrP^(Sc) can be used to carry out the basic assay of the presentinvention when the sample has been treated with dispase sufficiently tohydrolyze all or substantially all of the PrP^(C) present in the sample.Another useful antibody for binding to PrP^(C) is the monoclonalantibody 263K 3F4 produced by the hybridoma cell line ATCC HB9222deposited on Oct. 8, 1986 in the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852 and disclosed and described in U.S.Pat. No. 4,806,627 issued Feb. 21, 1989—incorporated by reference todisclose antibodies which selectively bind PrP^(c). The term “antibody”encompasses all types of antibodies, e.g. polyclonal, monoclonal, andthose produced by the phage display methodology. Particularly preferredantibodies of the invention are antibodies which have a relatively highdegree of affinity for both native PrP^(C) and treated PrP^(Sc) but arelatively low degree of or substantially no binding affinity forPrP^(Sc). More specifically, antibodies of the invention preferably havefour times or more, more preferably fifteen times or more, and stillmore preferably 30 times or more binding affinity for both nativePrP^(C) and denatured PrP^(Sc) as compared with the binding affinity fornative PrP^(Sc).

“Purified antibody” refers to that which is sufficiently free of otherproteins, carbohydrates, and lipids with which it is naturallyassociated. Such an antibody “preferentially binds” to a denatureddisease conformation of a protein such as the denatured β-sheetconformation of A4β or PrP^(Sc) protein (or an antigenic fragmentthereof), and does not substantially recognize or bind to otherantigenically unrelated molecules. A purified antibody of the inventionis preferably immunoreactive with and immunospecific for a specificspecies and more preferably immunospecific for native PrP^(C) and fordenatured forms of PrP^(C) and PrP^(Sc) or, alternatively, for native oruntreated PrP^(Sc).

“Antigenic fragment” of a protein (e.g., a PrP protein) is meant aportion of such a protein which is capable of binding an antibody.

By “binds specifically” is meant high avidity and/or high affinitybinding of an antibody to a specific polypeptide e.g., epitope of aprotein, e.g., denatured PrP^(Sc) or denatured A4β protein. Antibodybinding to its epitope on this specific polypeptide is preferablystronger than binding of the same antibody to any other epitope,particularly those which may be present in molecules in associationwith, or in the same sample, as the specific polypeptide of intereste.g., binds more strongly to epitope fragments of a protein such asPrP^(Sc) so that by adjusting binding conditions the antibody bindsalmost exclusively to an epitope site or fragments of a desired proteinsuch as an epitope fragment exposed by denaturing of PrP^(Sc) and notexposed on native PrP^(Sc).

By “detectably labeled antibody”, “detectably labeled anti-PrP” or“detectably labeled anti-PrP fragment” is meant an antibody (or antibodyfragment which retains binding specificity), having an attacheddetectable label. The detectable label is normally attached by chemicalconjugation, but where the label is a polypeptide, it couldalternatively be attached by genetic engineering techniques. Methods forproduction of detectably labeled proteins are well known in the art.Detectable labels known in the art, but normally are radioisotopes,fluorophores, paramagnetic labels, enzymes (e.g., horseradishperoxidase), or other moieties or compounds which either emit adetectable signal (e.g., radioactivity, fluorescence, color) or emit adetectable signal after exposure of the label to its substrate. Variousdetectable label/substrate pairs (e.g., horseradishperoxidase/diaminobenzidine, avidin/streptavidin, luciferase/luciferin),methods for labeling antibodies, and methods for using labeledantibodies are well known in the art (see, for example, Harlow and Lane,eds. (Antibodies: A Laboratory Manual (1988) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.)). Europium is a particularlypreferred label.

Abbreviations used herein include:

CNS for central nervous system;

BSE for bovine spongiform encephalopathy;

CJD for Creutzfeldt-Jacob Disease;

FFI for fatal familial insomnia;

GdnHCl for Guanidine hydrochloride;

GSS for Gerstamnn-Strassler-Scheinker Disease;

Hu for human;

HuPrP for human prion protein;

Mo for mouse;

MoPrP for mouse prion protein;

SHa for a Syrian hamster;

SHaPrP for a Syrian hamster prion protein;

Tg for transgenic;

Tg(SHaPrP) for a transgenic mouse containing the

PrP gene of a Syrian hamster;

Tg(HuPrP) for transgenic mice containing the complete human PrP gene;

Tg(ShePrP) for transgenic mice containing the complete sheep PrP gene;

Tg(BovPrP) for transgenic mice containing the complete cow PrP gene;

PrP^(Sc) for the scrapie isoform of the prion protein;

PrP^(C) for the cellular contained common, normal isoform of the prionprotein;

PrP 27-30 or PrP^(Sc) 27-30 for the treatment or protease resistant formof PrP^(Sc);

MoPrP^(Sc) for the scrapie isoform of the mouse prion protein;

MHu2M for a chimeric mouse/human PrP gene wherein a region of the mousePrP gene is replaced by a corresponding human sequence which differsfrom mouse PrP at 9 codons;

Tg(MHu2M) mice are transgenic mice of the invention which include thechimeric MHu2M gene;

MHu2 MPrP^(Sc) for the scrapie isoform of the chimeric human/mouse PrPgene;

PrP^(CJD) for the CJD isoform of a PrP protein;

Prnp^(0/0) for ablation of both alleles of an endogenous prion proteingene, e.g., the MoPrP gene;

Tg(SHaPrP^(+/0))81/Prnp^(0/0) for a particular line (81) of transgenicmice expressing SHaPrP, +/0 indicates heterozygous;

Tg(HuPrP)/Prnp^(0/0) for a hybrid mouse obtained by crossing a mousewith a human prion protein gene (HuPrP with a mouse with both alleles ofthe endogenous prion protein gene disrupted;

Tg(MHu2M)/Prnp^(0/0) for a hybrid mouse obtained by crossing a mousewith a chimeric prion protein gene (MHu2M) with a mouse with bothalleles of the endogenous prion protein gene disrupted;

TTR for transthyretin;

FVB for a standard inbred strain of mice often used in the production oftransgenic mice since eggs of FVB mice are relatively large and toleratemicroinjection of exogenous DNA relatively well;

[PrP_(β)]—concentration of prion protein in β-sheet conformation;

[βA4_(β)]—concentration of βA4 in β-sheet conformation;

[DRC]—concentration of a disease related conformation of a protein.

General Aspects of the Invention

The assay method comprises providing a sample suspected of containing aprotein which assumes a first conformation and a second disease relatedconformation and is capable of detecting a disease conformation of theprotein when present in a very low concentration relative to theconcentration of other proteins and compounds including the non-diseaseconformation.

The assay methods disclosed allows one to isolate and detect thepresence of a disease related conformation of a protein (e.g., PrP^(Sc))present in a sample also containing the non-disease related conformationof the protein (e.g., PrP^(C)). The sample is treated (e.g., contactedwith dispase) in a manner which hydrolyzes the PrP^(C) and not thePrP^(Sc). The hydrolyzation reaction is stopped (e.g., by the additionof EDTA). The treated sample is contacted with a binding partner (e.g.,a labeled antibody which binds PrP^(Sc)) and the occurrence of bindingprovides and indication that PrP^(Sc) is present. Alternatively thePrP^(Sc) of the treated sample is denatured (e.g., contacted withguanadine) or unfolded. The unfolded PrP^(Sc) is contacted with abinding partner (e.g., labeled 3F4) and the occurrence of bindingindicates the presence of PrP^(Sc) in the sample.

In accordance with any of the assay embodiments it is preferable topre-treat the sample being tested to (1) remove as many contaminantproteins as possible; and (2) increase the concentration of diseaserelated protein in the sample relative to the non-disease relatedconformation of the protein. For example, the initial sample can bechemically treated with a compound which preferentially degrades ordenatures contaminant proteins and/or the relaxed, non-disease form ofthe protein and/or is exposed to antibodies which preferentially bind to(in order to remove) contaminants and/or non-disease conformation of theprotein.

It may be possible to enhance further the sensitivity of various aspectsof the invention by concentrating the disease conformation of a proteinby adding a compound which selectively binds to the disease conformationto form a complex and centrifuging the sample to precipitate out thecomplex which is then tested in accordance with the methods describedhere. Specifics regarding such concentration methods are described indetail in our co-pending application Ser. No. 09/026,967 entitled“Process for Concentrating Protein with Disease-Related Conformation”now issued as U.S. Pat. No. 5,977,324 on Nov. 2, 1999.

The different embodiments of the assay of the invention described aboveare all “direct” types of immunoassays—meaning that the sample isdirectly assayed with the labeled antibody either with or withouttreatment to change the conformation of any disease related conformationproteins present in the sample. An “indirect” assay may also be used.For example, it may be desirable to enhance the number of diseaserelated proteins in the sample (if any) by the use of a transgenic mouseand thereby enhance any signal obtained. To carry out these embodimentsof the invention, the sample is first used to inoculate a transgenicmouse which has had its genome modified so that it will develop symptomsof disease when inoculated with proteins in the disease relatedconformation. After the mice are inoculated, a sufficient period of timeis allowed to pass (e.g., 30 days) after which the transgenic animal issacrificed and a sample such as homogenized brain tissue from the mouseis used in the direct assay described above. The present inventionenhances the ability of transgenic mice to detect prions by shorteningthe period of time which must pass until a determination can be made asto whether the original sample included proteins in the disease relatedconformation. It would also be possible to use mice of the typedisclosed and described in any of U.S. Pat. No. 5,565,186; 5,763,740; or5,792,901 or to apply epitope tagged PrP as disclosed in U.S. Pat. No.5,750,361 to affinity purify the PrP^(Sc) from the brain of a Tg mouseand thereafter apply the assay of the present invention. Without thepresent invention the mouse is inoculated and one must wait until theinoculated mouse actually demonstrates symptoms of the disease.Depending on the mouse, this can take several months or even years. Anyof the assays of the present invention could be used with any transgenicmice such as those described above. The assay could be used well beforethe mouse developed symptoms of disease thereby shortening the timeneeded to determine if a sample includes infectious proteins.

The assay methodology of the present invention can be applied to anytype of sample when the sample is suspected of containing a proteinwhich occurs in at least two conformations. The protein must occur inone conformation which binds to known antibodies, antibodies which canbe generated or other specific binding partners. The second conformationmust be sufficiently different from the first conformation in terms ofits ability to be hydrolyzed by compound (e.g., dispase). In itsconceptually simplest form, the invention works best when a compoundquickly and complete hydrolyzes the non-disease conformation of theprotein without affecting the disease related conformation. However, inreality, a given protein may have more than two conformations. Theprotein may have more than one non-disease conformation and more thanone disease related conformation, (Telling, et al., Science (1996)). Theinvention is still useful when multiple conformations of non-disease anddisease forms of the protein exist—provided that (1) at least onenon-disease conformation differs from at least one disease conformationin terms of its ability to be hydrolyzed by a compound.

As indicated above, the assay of the invention can be used to assay anytype of sample for any type of protein, provided the protein includes anon-disease and a disease related conformation. However, the inventionwas particularly developed to assay samples for the presence of (1) PrPproteins and determine whether the sample included a PrP protein in itsdisease conformation, i.e., included PrP^(Sc) (2) insoluble forms of PA4associated with Alzheimer's disease and (3) transthyretin. Accordingly,much of the following disclosure is directed to using the immunoassay ofthe present invention to detect the presence of either PrP^(Sc) (or to alesser degree βA4 or transthyretin (TTR)) in a sample—it beingunderstood that the same general concepts are applicable to detectingdisease related conformations of a wide range of different types ofproteins.

Europium labeled antibodies used (3F4) have a high binding affinity forPrP^(C) (non-disease conformation) which comprises an α-helical richconformation. The antibodies have a low binding affinity for PrP^(Sc)(disease conformation) which comprises a β-sheet rich conformation. TheIgG may be obtained from common monoclonal, polyclonal, or recombinantantibodies, typically recognizing the sequence 90-145 of PrP^(C) andconformationally unfolded prion protein. Different conformations ofrecombinant prion protein were chemically crosslinked to polystyreneplates through a glutaraldehyde activation step. The relative affinitiesof the Eu-labeled IgG with α-helical, β-sheet, and random coilconformation of recombinant Syrian hamster prion protein correspondingto sequence 90-231 were determined by time-resolved,dissociation-enhanced fluorescence in a 96-well polystyrene plateformat.

After the labeled antibodies have been provided with sufficient time,temperature and chemical conditions (e.g., pH) to bind to theappropriate proteins present in the respective portions the level ofbinding of the labeled antibody to protein is determined.

Once a labeled antibody has bound to its target detection may bedifficult due to the low concentration of the target molecule in thesample. Different procedures can be used for detection.

Time-resolved, dissociation-enhanced fluorescence and more preferablydual wavelength, laser-driven fluorometers are particularly usefuldevices—see Hemmilä) et al., Boianalytical Applications of LabelingTechnologies (eds. Hemmila) 113-119 (Wallas Oy. Turku, Finland, 1995).

These devices make it possible to detect concentrations in an amount inthe range of about 1×10³ particles per ml or less. A high degree ofsensitivity is preferred because in most samples the concentration ofprotein in the disease conformation will be very low. For example, thenon-disease conformation of the protein might be present in an amount ofabout 1×10⁸ particles/ml while the disease conformation of the proteinis only present in an amount of 1×10⁴ particles/ml.

The assay can be used to test for the presence of the diseaseconformation of a given protein within any type of sample. Some of themost typical samples to be tested include pharmaceuticals which includecomponents which are derived from living mammals or use materialsderived from living mammals in their processing. It would also bedesirable to test organs for transplantation and food items such as beefwhich was suspected of containing infectious prions. The invention couldbe used for testing for the presence of the disease conformation of oneor more types of proteins such as infectious PrP^(Sc) inpharmaceuticals, cosmetics, biopsy or autopsy tissue, brain, spinalcord, peripheral nerve, muscle, cerebrospinal fluid, blood and bloodcomponents, lymph nodes, and in animal or human-derived culturesinfected or potentially infected by disease forms of proteins such asprions. The brains of cows suspected of being infected with prions(i.e., BoPrP^(Sc)) could be tested to determine if the cows can besafely used for human consumption.

Treatment—General

An assay of the invention can use all or any of three basic types oftreatment which are defined above. The treatments are (1) pretreatment,(2) unfolding treatment and (3) hydrolysis treatment. In general theconditions for pretreatment are gentle, those for unfolding treatmentmoderate and those for hydrolysis treatment are harsh. Each type oftreatment can employ the same means (e.g. proteases, time, pH,temperature, etc.) but employs each to a different degree, e.g. higherconcentration, longer time, higher temperature. However, the hydrolysistreatment must employ a compound which selectively hydrolyzes only thenon-disease conformation and not the disease conformation.

Pretreatment

Before carrying out treatment or antibody testing of the sample it maybe desirable to subject the sample to pretreatment. The pretreatment iscarried out in order to destroy or remove unrelated proteins as well assome of the non-disease form of the protein present within the sample.Examples of pretreatment methodology include producing a column whichincludes antibodies bound to support surfaces which antibodies bind tothe non-disease conformation of the protein thereby removing as much ofthe non-disease conformation of the proteins possible. Antibodies whichbind unrelated but common proteins can also be used. Alternatively, thesample can be subjected to physical treatment such as long termhydrostatic pressure or temperature alone or in combination withchemicals such as acids or alkalines as indicated above to destroyproteins present in the sample which proteins are not related to thosebeing assayed for or are in the non-disease conformation. In someinstances proteins in the non-disease and disease conformation will bedestroyed. However, a higher relative percentage of the proteins in thenon-disease conformation will be destroyed because these proteins areinitially in a looser conformation which is more vulnerable todestruction. Thus, the pretreatment methodology results in a samplewhich includes a relatively lower concentration of the non-diseaseconformation of the protein relative to the concentration of the diseaseconformation of the protein. Further, the pretreated sample will have alower concentration of unrelated proteins. This increases thesensitivity of the assay making it possible to detect lowerconcentrations of the disease conformation of the protein. Removal ofproteins is preferred over destruction of such in that destruction willdecrease sensitivity if the disease conformation is destroyed. Aparticularly useful pretreatment method is disclosed in U.S. Pat. No.5,977,324, issued Nov. 2, 1999.

Unfolding Treatment

The unfolding treatment denatures the protein but does not hydrolyzeproteins of interest and can include exposing the proteins to anyphysical and/or chemical means which causes the protein which isoriginally present in a tightened, disease related conformation (e.g.,PrP^(Sc)) to assume a more relaxed conformation which has a higherdegree of binding affinity for any binding partner such as antibodies(e.g., expose an N-terminal epitope of PrP^(Sc)). In general, theunfolding treatment involves subjecting the protein to some means whichcauses epitopes on the protein which were not previously exposed orpartially exposed to become exposed or become more exposed so that anantibody or other binding partner can more readily bind to the newlyexposed epitope.

Methods used for unfolding treatment may include: (1) physical, such ashydrostatic pressure or temperature, (2) chemical, such as acidic oralkaline pH, chaotropic salts, denaturing detergents, guanidinehydrochloride and proteinases such as Proteinase K and (3) combinationsof above.

The treatment time will vary depending on the treatment used but shouldbe carried out for sufficient time to obtain the desired effect, e.g.for unfolding treatment to expose new binding sites but not so long asto completely denature or hydrolyze the protein. When carrying outunfolding treatment on PrP proteins without chemical treatment thetemperature is raised to about 40° C. to about 80° C. for a timesufficient to obtain the desired amount of unfolding of PrP^(Sc). Thetemperature can be lower and the time shorter if the pH is raised to 12or 13.

Hydrolysis Treatment

The hydrolysis treatment is a lytic treatment which is the mostimportant treatment method used in one embodiment of the assays of theinvention. After a sample has been subjected to the pretreatmenttreatment it is subjected to the hydrolysis treatment. This treatmentwill destroy or hydrolyze all or substantially all protein in the samplewhich is in the non-disease conformation and not hydrolyze the proteinin the disease conformation. The hydrolysis treatment is prferably viaan enzyme such as a hydrolase that acts on peptide bonds, preferably aneutral protease, more preferably a metalloendopeptidase, and mostpreferably dispase or leucostoma peptidase A. The proteases used in themethod of the invention may be used alone, in combination, or inconjunction with enzymes having similar but distinct activity such as acarbohydrase, e.g. collagenase, amylase, or alkaline serine protease.The concentration of the treating compounds as well as the time andtemperature will vary with the protein being treated and end result tobe obtained. For example, with PrP the treatment is carried out tohydrolyze all or substantially all non-PrP^(C) present, but nothydrolyze PrP^(Sc) present. The object of this treatment is to hydrolyzeas much non-disease protein as possible (preferably all) whilehydrolyzing as little (preferably none) disease related protein aspossible. The treatment is preferably designed such that it can bequickly and completely stopped at any given time. For example,hydrolysis of PrP^(C) with dispase or other related proteases can bestopped by adding EDTA.

The following list of enzymes are preferred compounds of the method ofthe invention: Enzyme Biological Source Dispase Bacillus polymyxaAtrolysin A, B, C, Western diamondback rattlesnake Crotalus E and Fatrox Envelysin Various member of the class Echinoidea Thimetoligopeptidase Related to Saccharolysin from Saccharomyces cerevisiae.Matrilysin Ratuterus Vibriolysin Vibrio proteolyticus (formerlyAeromonas proteolytica) Coccolysin Streptococcus thermophilus MycolysinStreptomyces griseus Meprin A ratus and mus kidney and intestinal brushborder Astacin The crayfish Astacus fluviatilis AureolysinStaphylococcus aureus Leishmanolysin Various species of Leishmaniaprotozoans Peptidyl-Asp Psuedomonas fragi Metalloendopeptidase AutolysinChlamydomonas reinhardtii Deuterolysin Penicillium roqueforti; speciesvariants include Penicillium caseicolum, Aspergillus sojae, andAspergillus oryzae. Bothrolysin Venom of jararaca snake Bothropsjararaca Stromelysin 1 and 2 Human rheumatoid synovial fibroblastsBacillolysin Bacillus subtilis; species variants include Bacillusamyloliquefaciens, Bacillus megaterium, Bacillus mesentericus, Bacilluscereus, and Bacillus stearothermophilus Thermolysin Bacillusthermoproteolyticus; species variants include Micrococcus caseolyticusand Aspergillus oryzae. Aeromonolysin Honey fungus Armillaria melleaLeucolysin Venom of the western cottonmouth moccasin snake, Agkistrodonpiscivorus Mycolysin Streptomyces griseus, Streptomyces naraensis, andStreptomyces cacaoi Pseudolysin Psuedomonas aeruginosa Peptidyl-LysPseudomonas fragi Metalloendopeptidase Aureolysin Staphylococcus aureusNeprilysin Widely distributed in mammal tissues, including brain, liverand lung; abundant in kidney brush border membrane β-lytic Achromobacterlyticus and Lysobacter Metalloendopeptidase enzymogenes Peptidyl-AspPseudomonas fragi Metalloendopeptidase Ophiolysin Venom of the KingCobra Ophiophagus hannah. Pitrilysin Escheria coli Insulysin mammals andDrosophila melanogasters Serralysin Pseudomonas aeruginosa; speciesvariants include Escheria freundii, Serratia marcescens, and Erwiniachrysanthemi.

The method of the invention is not limited to these enzymes, and thusother enzymes predicted by those skilled in the art to function in themethod of the invention may be used.

Binding Proteins to Support Surfaces

The method of chemical or affinity coupling of PrP protein to theplastic support are generally described in available literature and mayvary. The antibodies used in the diagnostic assay are polyclonal,monoclonal or recombinant Fab and need to be species specific withpreferential binding to the native PrP^(C) or denatured form of PrP^(Sc)with preferably at least 4-fold lower reactivity with infectiousPrP^(Sc), assuming the same amount of the antigen.

Using the Assay to Detect Prions (PrP^(Sc))

One aspect of the invention is a two step process to diagnose priondisease by quantitatively measuring the native infectious form ofPrP^(Sc) protein in sample material or in the brains of susceptibleanimals inoculated with such material. The sample is preferablypretreated to remove as much unrelated and non-disease protein aspossible. The pretreated sample is first subjected to hydrolysistreatment and then crosslinked to the plastic support.

To measure the concentration of PrP^(Sc) when it is much less thanPrP^(C), the detection system has to have extreme sensitivity and alinear range of at least 10⁴. The assay described herein can readilydetect PrP^(Sc) at a concentration of (approximately) 50 pg/ml usingEuropium-labeled IgG. Assuming 10⁵-10⁶ PrP^(Sc) molecules per ID₅₀ unitthe present assay can readily detect 5×10²-5×10³ ID₅₀ units per ml.

The assay can detect PrP^(Sc) in mixtures (by direct method) where theconcentration of PrP^(Sc) is less than 1% of the concentration ofPrP^(C). Additional sensitivity can be achieved by immunoprecipitation,using a sandwich format for a solid state assay, differentialcentrifugation with detergent extraction to remove PrP^(C), the indirecttransgenic animal method or combinations of these methods. Aconservative estimate is that such procedures should allow measurementof between 5 and 50 ID₅₀ units per ml or less conservatively to measurebetween 0.1 and 0.01 ID₅₀ units per ml. Such measurements would providea rapid, “positive” means of establishing biological sterility which isthe “absence” of infectivity.

Antibodies

Method of generating antibodies are generally known to those skilled inthe art. In that the disease form is often in a tighter configurationthan the non-disease form, with less epitopes exposed, one can readilygenerate antibodies which bind only to the non-disease form of theprotein or the treated disease form. For example, antibodies detectingtreated forms of PrP^(Sc) protein and PrP^(C) protein may be generatedby immunizing rabbits or mice with α-helical conformations ofrecombinant PrP, native PrP^(C) from animal brains, synthetic peptidesin α-helical or random coil conformations, or against denatured PrP^(Sc)or PrP 27-30. Only antibodies with affinity at least 4 fold higher forPrP^(C) (or denatured conformation of PrP^(Sc) of the same species) ascompared to their affinity for PrP^(Sc) should be selected. The methodof antibody generation, purification, labeling and detection may vary.

The IgG or Fab's may be purified from different sources by affinity HPLCusing protein A column and Size exclusion HPLC. The purified antibodiesmay be labeled with Europium and detected by time resolved fluorescence.The antibody binding to different conformations of PrP protein may bemeasured by time-resolved, dissociation-enhanced fluorescence. However,the system of detection of PrP-bound IgG on solid support in situ or insolution may vary. Further, it is possible to use direct or indirectimmunological methods including direct radiolabels, fluorescence,luminescence, avidin-biotin amplification, or enzyme-linked assays withcolor or luminescent substrates.

An antibody which can be used in the invention is disclosed in U.S. Pat.No. 4,806,627, issued Feb. 21, 1989, disclosing monoclonal antibody 263K3F4, produced by cell line ATCC HB9222 deposited on Oct. 8, 1986, whichis incorporated herein by reference. The cell line producing theantibody can be obtained from the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md. 20852.

In general, scrapie infection fails to produce an immune response, withhost organisms being tolerant to PrP^(Sc) from the same species.Antibodies which bind to either PrP^(c) or PrP^(Sc) are disclosed inWO97/10505, published Mar. 20, 1997. Any antibody binding to PrP^(c) andnot to PrP can be used, and those skilled in the art can generate suchusing known procedures, e.g., see methods of producing page displayantibody libraries in U.S. Pat. No. 5,223,409. Polyclonal anti-PrPantibodies have though been raised in rabbits following immunizationwith large amounts of formic acid or SDS-denatured SHaPrP 27-30[Bendheim, Barry et al. (1984) Nature 310:418-421; Bode, Pocchiari etal. (1985) J Gen Virol 66:2471-2478; Safar, Ceroni et al. (1990)Neurology 40:513-517]. Similarly, a handful of anti-PrP monoclonalantibodies against PrP 27-30 have been produced in mice [Barry andPrusiner (1986) J Infect Dis 154:518-521; Kascsak, Rubenstein et al.(1987) J Virol 61:3688-3693]. These antibodies were generated againstformic acid- or SDS-denatured PrP 27-30 and are able to recognize nativePrP^(C) and treated or denatured PrP^(Sc) from both SHa and humansequally well, but do not bind to MoPrP. Not surprisingly, the epitopesof these antibodies were mapped to regions of the sequence containingamino acid differences between SHa- and MoPrP [Rogers, Yehiely et al.(1993) Proc Natl Acad Sci USA 90:3182-3186].

It is not entirely clear why many antibodies of the type described inthe above cited publications will bind to PrP^(C) and treated ordenatured PrP^(Sc) but not to native PrP^(Sc). Without being bound toany particular theory it is suggested that such may take place becauseepitopes which are exposed when the protein is in the PrP^(C)conformation are unexposed or partially hidden in the PrP^(Sc)configuration—where the protein is relatively insoluble and morecompactly folded together.

For purposes of the invention an indication that no binding occurs meansthat the equilibrium or affinity constant K_(a) is 10⁶ l/mole or less.Further, binding will be recognized as existing when the K_(a) is at 10⁷l/mole or greater, preferably 10⁸ l/mole or greater. The bindingaffinity of 10⁷ l/mole or more may be due to (1) a single monoclonalantibody (i.e., large numbers of one kind of antibodies) or (2) aplurality of different monoclonal antibodies (e.g., large numbers ofeach of five different monoclonal antibodies) or (3) large numbers ofpolyclonal antibodies. It is also possible to use combinations of(1)-(3). Selected preferred antibodies will bind at least 4-fold moreavidly to the treated or denatured PrP^(Sc) forms of the protein whencompared with their binding to the native conformation of PrP^(Sc). Thefour fold differential in binding affinity may be accomplished by usingseveral different antibodies as per (1)-(3) above and as such some ofthe antibodies in a mixture could have less than a four fold difference.

A variety of different types of assays of the invention may be used withone or more different antibodies. Those skill in the art will recognizethat antibodies may be labeled with known labels and used with currentlyavailable robotics, sandwich assays, electronic detectors, flowcytometry, and the like.

Diseases Associated with Insoluble Proteins

Much of the disclosure and the specific examples provided herein relateto the use of the assay in connection with determining the presence ofPrP^(Sc) in the sample. However, as indicated above, the assay of theinvention can be applied to determining the presence of any proteinwhich assumes two different conformational shapes, one of which isassociated with the disease. The following is a non-limiting list ofdiseases with associated insoluble proteins which assume two or moredifferent conformations. Disease Insoluble Proteins Alzheimer's DiseaseAPP, Aβ peptide, α1-antichymotrypsin, tan, non-Aβ component Priondiseases, PrP^(Sc) Creutzfeld Jakob disease, scrapie and bovinespongeform encephalopathy ALS SOD and neurofilament Pick's disease Pickbody Parkinson's disease Lewy body Diabetes Type 1 Amylin Multiplemyeloma-- IgGL-chain plasma cell dyscrasias Familial amyloidoticTransthyretin polyneuropathy Medullary carcinoma Procalcitonin ofthyroid Chronic renal failure β₂--microglobulin Congestive heart failureAtrial natriuretic factor Senile cardiac and Transthyretin systemicamyloidosis Chronic inflammation Serum amyloid A Atherosclerosis ApoA1Familial amyloidosis Gelsolin

It should be noted that the insoluble proteins listed above each includea number of variants or mutations which result in different strainswhich are all encompassed by the present invention. Known pathogenicmutations and polymorphisms in the PrP gene related to prion diseasesare given below and the sequences of human, sheep and bovine are givenin U.S. Pat. No. 5,565,186, issued Oct. 15, 1996. MUTATION TABLEPathogenic human Human Sheep Bovine mutations PolymorphismsPolymorphisms Polymorphisms 2 octarepeat insert Codon 129 Codon 171 5 or6 octarepeats Met/Val Arg/Glu 4 octarepeat insert Codon 219 Codon 136Glu/Lys Ala/Val 5 octarepeat insert 6 octarepeat insert 7 octarepeatinsert 8 octarepeat insert 9 octarepeat insert Codon 102 Pro-Leu Codon105 Pro-Leu Codon 117 Ala-Val Codon 145 Stop Codon 178 Asp-Asn Codon 180Val-Ile Codon 198 Phe-Ser Codon 200 Glu-Lys Codon 210 Val-Ile Codon 217Asn-Arg Codon 232 Met-Ala

It should also be noted that such proteins have two different3-dimensional conformations with the same amino acid sequence. Oneconformation is associated with disease characteristics and is generallyinsoluble whereas the other conformation is not associated with diseasecharacteristics and is soluble. The methodology of the present inventionis not limited to the diseases, proteins and strains listed.

Detecting the β-Sheet Form of βA4

One aspect of the invention involves a two step process to diagnoseAlzheimer's disease based on the presence of a constricted form of aprotein (βA4 amyloidosis) by quantitatively measuring β-sheet form ofβA4 protein in sample material, e.g., in the brain or body fluids. Thesample is divided into two aliquots. The first aliquot is crosslinked toa solid plastic (long chain polymeric material) support in nativeconformation through a chemical activation step under the nondenaturingconditions. The second portion of the sample is first subjected tounfolding treatment and then crosslinked to the plastic support. Bothportions of the sample material react in situ with the labeledantibodies that preferentially recognize soluble βA4 or unfoldingtreatment βA4 of the human or a given animal species. The amount of theantibody bound to unfolded or native conformations of βA4 protein isrecorded by the signal of the labeled secondary antibody. The excess ofthe signal obtained with the unfolding treated sample compared to thatexpected change in the signal obtained with the native α-helicalconformation of βA4 protein is the measure of the amount of β-sheetstructured βA4 in the original sample. The formula developed forcalculation of βA4 content is provided above in connection with thecalculation of PrP^(Sc) content.

The diagnosis of βA4 amyloidosis (Alzheimer's disease) is established bythree procedures: (1) measurement of denatured sample alone and bydetecting the increase in the total βA4 amount (concentration) in theexamined sample above the background levels of soluble βA4 obtained fromnormal controls; (2) calculation of the ratio between unfolding treatedversus native signal for a given antibodies (protein index)—for examplevalues higher than 2 for monoclonal antibody 6F3D and europium labeledsecondary antibody; (3) evaluation of the change of the denatured samplesignal over that expected change in the signal for α-helicalconformation of βA4 as a measure of the amount of infectious β-sheetstructured βA4 in the original sample. The formula developed forcalculation of βA4 content is provided above. The particular strain ofβA4 can also be determined using the same methodology described above todetermine the strain of PrP^(Sc) in a sample.

The invention provides a direct diagnostic method for detecting thepresence pathogenic forms of βA4 protein in pharmaceuticals, biopsy orautopsy tissue, brain, spinal cord, peripheral nerves, muscle,cerebrospinal fluid, blood and blood components, lymph nodes, and inanimal- or human-derived cultures expressing or potentially expressingβA4 protein. The invention also makes it possible to follow theα-helix-to-β-sheet conformational transition of βA4 protein, or itsfragments of synthetic or recombinant origin, and to provide a method toscreen compounds for their ability to stabilize the normal solubleconformation of βA4 protein and thus prevent conversion into pathogenicinsoluble and β-sheet-structured βA4 protein.

Typical methods of sample denaturation include: (1) physical, such ashydrostatic pressure or temperature, (2) chemical, such as acidic oralkaline pH, chaotropic salts, or denaturing detergents, and (3)combination of above. Methods of chemical or affinity coupling of βA4protein to a plastic support are described in available literature andmay vary. Antibodies used in the diagnostic assay may be polyclonal,monoclonal or recombinant Fab and must be species specific withpreferential binding to the soluble or denatured form of βA4 withpreferably at least a 2-fold difference in reactivity between α-helicaland β-sheet structured βA4, assuming the same amount of antigen.

Methods of sample attachment to the plastic support may vary and may becovalent or non-covalent as described in available literature. Thesensitivity of the assay described in the examples may be increased byusing high-affinity antibodies, sandwich formate, immunoprecipitation,or differential centrifugation. However, only the antibodies with anaffinity at least a 2 fold for unfolding treated as compared to thenative β-sheet conformation of βA4 of the same species shall be used forthe diagnostic assay. Methods of antibody generation, purification,labeling and detection may vary. The antibody binding to differentconformations of βA4 protein was measured by time-resolved,dissociation-enhanced fluorescence. However, the system of detection ofβA4-bound IgG on solid support in situ or in solution may vary and mayuse direct or indirect immunological methods including directradiolabels, fluorescence, luminescence, avidin-biotin amplification, orenzyme-linked assays with color or luminescent substrates.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use assays of the present invention, and are not intended tolimit the scope of what the inventors regard as their invention, nor arethey intended to represent or imply that the experiments below are allof or the only experiments performed. Efforts have been made to ensureaccuracy with respect to numbers used (e.g. amounts, temperature, etc.)but some experimental errors and deviations should be accounted for.Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degreescentigrade, and pressure is at or near atmospheric.

Example 1 Detection of PrP^(Sc) in Hamster Brain

To determine the levels of PrP^(Sc) in affected hamsters, a prioninfected hamster and a normal hamster were each sacrificed and theirbrains removed. A 10% (w/v) homogenate of each of the brains wasprepared by dispersing the brain tissue in PBS. The brain homogenate wasthen subjected to a low speed centrifugation of 500×g for 15 minutes toseparate the suspended proteins from unwanted cellular debris. The totalprotein concentration of the supernatant (S1) was measured using a BCAProtein Assay (Pierce) and the concentration of each brain homogenatewas adjusted with PBS to 3.5 mg/ml. A portion of the homogenate wassaved to serve as a control of total brain proteins.

The metalloendopeptidase dispase (Worthington) was added to theremainder of each sample at an enzyme to protein ratio of 1:35. Thehomogenates were digested with 100 μg/ml dispase for 60 min at 37° C. inthe presence of either 0.15% Zwittergent 3-12 (Calbiochem), or 0.2% or2% of sodium dodecyl sarcosinate (Sarkosyl). A sample without thedispase was also placed at 37° C. to serve as a digestion control.Following digestion the dispase was inactivated by the addition of 50 mMEDTA.

As a control, Proteinase K digestion was done on each untreated S1sample with 20 μg/ml at a ratio of enzyme to protein of 1:50 (Bolton,1982) in the presence of either 0.15% Zwittergent, 0.2% or 2% Sarkosyl.These reactions were terminated by the addition of 2 mM PMSF.

The digested samples were centrifuged at 100,000×g for 1 hour. Thepellet containing all the insoluble proteins was resuspended in aminimal volume of PBS and 0.15% Zwittergent. Subsequently, both thedigested samples and the whole brain homogenate samples were sonicatedin a water bath sonicator for 20 minutes to denature the proteinremaining after digestion.

Each sample was then analyzed for PrP protein content by immunoblot. A100 μl aliquot of each sample was placed in a 1.5 ml eppendorf tube withan equal volume of sample loading buffer (1X=50 mM Tris Cl, pH 6.8; 100mM DTT; 2% SDS; 0.1% bromophenol blue; and 10% glycerol). In addition,an aliquot of b-mercaptoethanol can be added to ensure denaturation ofthe proteins on the gel. The samples were run on a 10% polyacrylamidegel, molar ratio of bisacrylamide:acrylamide of 1:29, for 15 V/cm forabout 4 hours. The samples were each boiled for 10 minutes, and 15 μl ofeach sample was loaded onto the gel. For a more detailed description ofprotein separation via PAGE, see Schägger and Von Jagow, Nature166:368-379 (1987) and Laemmli, U.K. (1970) Nature 227, 680-685, whichare both incorporated herein in their entirety.

The gel was removed from the PAGE apparatus, and transferred ontouncharged nylon (Amersham) using an electroblotting apparatus. The geland the nylon were sandwiched between pieces of Whatman 3MM paper soakedin a transfer buffer containing Tris, glycine, SDS and methanol. Thesandwich was placed between graphite plate electrodes, with the nylon onthe anodic side. A current of 0.65 mA/sq. cm was applied for 1.5-2hours. Following transfer, the gel was stained with coomassie blue to besure the transfer was complete.

The nylon filter was placed in a heat-sealable plastic bag, and 0.1 mlof blocking solution were added per square cm of filter. The blockingsolution contained 5% (w/v) nonfat dry milk (Carnation); 0.01% antifoamA; and 0.02% sodium azide in PBS. After 1 hour shaking at roomtemperature, the monoclonal antibody 3F4 was added in a 1:100 dilution,and the filter incubated for 2-4 hours at 4° C. with gentle agitation ona platform shaker. Following incubation, the blocking solution andantibody were removed, and the filter washed three times.

The filter was incubated with an anti-Ig secondary antibody in blockingsolution for 1-2 hours at room temperature. The secondary antibody wasradiolabeled to allow immunoblot detection. For the radiolabeled I¹²⁵probe, approximately 10⁴ cpm of the reagent was added per squarecentimeter of filter. After incubation, the filter was washed severaltimes in PBS, each wash being about 10 minutes in length. The filter wasplaced in a cassette with a piece of Xomat X-ray film (Kodak) at −70° C.

The results of the immunoblot were as follows:

lane 1 normal hamster S1

Result: strong band at about 33-35 Kd

lane 2 normal hamster S1 at 37° C.

Result: strong band at about 33-35 Kd

lane 3 normal hamster S1 digested with 100 μg/ml dispase, 0.15%Zwittergent

Result: no band

lane 4 normal hamster S1 digested with 100 μg/ml dispase, 0.2% Sarkosyl

Result: no band

lane 5 normal hamster S1 digested with 100 μg/ml dispase, 2% Sarkosyl

Result: no band

lane 6 normal hamster S1 digested with 20 μg/ml protein kinase, 0.2%Sarkosyl

Result: no band

lane 7 normal hamster S1 digested with 20 μg/ml protein kinase, 2%Sarkosyl

Result: no band

lane 8 prion-infected hamster S1

Result: strong band at about 33-35 Kd

lane 9 prion-infected hamster S1 at 37° C.

Result: strong band at about 33-35 Kd

lane 10 prion-infected hamster S1 digested with 100 μg/ml dispase, 0.15%Zwittergent

Result: very strong band at about 33-35 Kd

lane 11 prion-infected hamster S1 digested with 100 μg/ml dispase, 0.2%Sarkosyl

Result: very strong band at about 33-35 Kd

lane 12 prion-infected hamster S1 digested with 100 μg/ml dispase, 2%Sarkosyl

Result: very strong band at about 33-35 Kd

lane 13 prion-infected hamster S1 digested with 20 μg/ml protein kinase,0.2% Sarkosyl

Result: very strong band at about 27-30 Kd

lane 14 prion-infected hamster S1 digested with 20 μg/ml protein kinase,2% Sarkosyl

Result: very strong band at about 27-30 Kd

These results showed that normal hamster brain PrP^(C) was not detectedafter digestion with Dispase or Proteinase K, while samples from theprion infected brain showed a very strong signal of protease resistantprotein. The prion infected samples digested with proteinase K showed anexpected shift in the size of the molecular weight corresponding to thedigestion of the n-terminus of the protein. The digestion with Dispaseshowed no shifting in the molecular weight. This finding showed that theDispase is selective for the normal conformation of the protein.

Example 2 Detection of PrP^(Sc) in Mouse Brain

To determine the levels of PrP^(Sc) in affected mice, a prion infectedmouse and a normal mouse are each sacrificed and their brains removed. A10% (w/v) brain homogenate from normal and prion infected mice isprepared in PBS. After a low speed centrifugation at 500×g for 15 min,the total protein in the supernatant (S1) is measured usingspectrophotometric assays, and the concentration is adjusted to 2.5mg/ml with PBS.

The samples are digested with 500 U/ml Leucolysin for 45 min at 37° C.in the presence of 2% Sarkosyl. The digestion is stopped by the additionof 50 mM EDTA. An aliquot of the proteins obtained in S1 both before andafter the leucolysin digestion are electrophoresed at 4° C. on an 8%polyacrylamide slab gel as described in the Laemmli reference but in theabsence of SDS and 2-mercaptoethanol. This allows the nondenaturedproteins to migrate through the polyacrylamide while preserving thenative structure of the protein. Once immobilized in the polyacrylamide,the proteins of the gel are then transferred to nitrocellulose forprotein detection. Transfer may occur as in Example 1, or a semi-drytransfer apparatus may be used (Reference Maniatis).

The digested and undigested sample of both infected and normal mouse aredetected in Western blot as described in Example 1, but using amonoclonal antibody that recognizes the native PrP^(Sc) form of theprotein. Such antibodies are described in U.S. Ser. No. 08/804,536 nowissued as U.S. Pat. No. 5,891,641 on Apr. 6, 1999, Ser. Nos. 09/026,957and 09/151,057, each of which is incorporated herein by reference intheir entirety. Preferably, the antibody used is the PrP^(Sc)-specificantibody R1. The antibodies are added at a concentration of about 1:100to 1:200, depending on the antibody used and the amount of proteinpredicted to be immobilized on the nitrocellulose.

The nitrocellulose is placed in a heat-sealable plastic bag, and 0.1 mlof blocking solution is added per square cm of filter. The blockingsolution contains 5% (w/v) nonfat dry milk (Carnation); 0.01% antifoamA; and 0.02% sodium azide in PBS, and Tween 20 added to a finalconcentration of 0.02%. The Tween 20 is a gentle detergent that furtheraids in reducing the background. After 1 hour shaking at roomtemperature, the R1 antibody is added in a 1:100 dilution, and thefilter is incubated for 2-4 hours at 4° C. with gentle agitation on aplatform shaker. Following incubation, the blocking solution andantibody are removed, and the filter is washed three times.

The filter is incubated with an anti-Ig secondary antibody in blockingsolution for 1-2 hours at room temperature. The secondary antibody isenzyme-conjugated with horseradish peroxidase to allow immunoblotdetection. The secondary antibody is added at a much more dilute levelthan the primary antibody, from 1:500 to 1:2000 dilution. Afterincubation, the filter is washed several times in PBS, each wash beingabout 10 minutes in length. The filter is then placed in a cassette witha piece of Xomat X-ray film (Kodak) at −70° C.

The resulting blot will have a band corresponding to PrP^(Sc) in thesample of the treated, prion infected mouse brain, both before and afterdigestion with Leucolysin. The sample of the normal mouse brain proteinwill show a low level band due to background binding of the PrP^(Sc).The lane with the normal brain sample subjected to hydrolysis withLeucolysin, however, will not have a detectable band, since theLeucolysin will have hydrolysed the PrP protein in the sample.

Example 3 Detection of PrP^(Sc) in Cow Brain

A 10% (w/v) brain homogenate from normal and prion infected cows isresuspended in 1 L of 25 mM Tris·HCl, pH 8.0, 5 mM EDTA (buffer A). Thisis centrifuged at 10,000×g for 20 min, and the supernatant containingsoluble periplasmic proteins is discarded. The pellet is resuspended in1 L of buffer A, passed through a cell disrupter twice (MicrofluidicsInternational, model MF110), and centrifuged at 30,000×g for 1 h, afterwhich the supernatant is discarded and the pellet is washed once inbuffer A and centrifuged again at 30,000×g for 1 hour. At this stage thepellet could be stored at −20° C. prior to hydrolysis.

The β-Lytic Metalloendopeptidase digestion is done at an enzyme toprotein ratio of 1:40. The protein is digested with 100 μg/ml β-LyticMetalloendopeptidase (Sigma) for 75 min at 40° C. in a buffered pH 8.0solution containing 0.2% Sarkosyl. The digestion is stopped by theaddition of 50 mM EDTA. Following the hydrolysis of the PrP^(C)conformation of the prion protein, the PrP^(Sc) can be denatured toallow the 3F4 antibody to recognize its epitope. Both the normal andprion infected samples are treated with a denaturation solution of 6Mguanidine HCl. The denaturation solution is prepared by diluting 10×buffer (250 mM HEPES (pH 7.9); 30 mM MgCl₂; 40 mM KCl) with five volumesof distilled water. An appropriate amount of the guanidine HCl is added,and the solution is brought to 1× using distilled water. Finally,dithiothreitol is added to a final concentration of 1 mM. The hydrolyzedsamples are subjected to treatment with the denaturation solution for 30minutes at 4° C.

The samples are then loaded onto 10% polyacrylamide gels as described inExample 1, and transferred to nylon membrane. The nylon membrane isplaced in a heat-sealable plastic bag, and 0.1 ml of blocking solutionis added per square cm of filter. The blocking solution contains 5%(w/v) nonfat dry milk (Carnation) and 0.02% sodium azide in PBS. After 1hour shaking at room temperature, the monoclonal antibody 3F4 is addedin a 1:200 dilution, and the filter incubated for 2-4 hours at 4° C.with gentle agitation on a platform shaker. Following incubation, theblocking solution and antibody are removed, and the filter washed threetimes. The filter is incubated with an anti-Ig secondary antibody inblocking solution for 1-2 hours at room temperature, the filter washedseveral times in PBS, and placed in a cassette with a piece of XomatX-ray film (Kodak) at −70° C.

The determination of prion infection is based on a comparison ofrecognition of PrP in the normal and infected samples. The lanescontaining unhydrolyzed S1 should contain relatively similar amounts ofPrP protein recognized by the 3F4 antibody. The lanes containing thehydrolyzed, guanidine HCl-treated samples will allow the detection ofPrP^(Sc) in the infected sample, since the only form left after thehydrolysis is the PrP^(Sc) form. The ratio of signal between thehydrolyzed and unhydrolyzed sample from the infected cow will determinethe percentage of PrP that is in the PrP^(Sc) conformation. Thehydrolyzed normal cow sample will further serve as a control that thehydrolysis of the PrP^(C) conformation was complete.

Example 4 Comparison of 3F4 and R1 Staining of a Sample

To determine the levels of PrP^(Sc) in affected mice, a prion infectedmouse and a normal mouse are each sacrificed and their brains removed. A10% (w/v) homogenate of each of the brains was prepared by dispersingthe brain tissue in PBS. The brain homogenate was then subjected to alow speed centrifugation of 500×g for 15 minutes to separate thesuspended proteins from unwanted cellular debris. The total proteinconcentration of the supernatant (S1) was measured using a BCA ProteinAssay (Pierce) and the concentration of each brain homogenate wasadjusted with PBS to 3.5 mg/ml. A portion of the homogenate was saved toserve as a control of total brain proteins.

Each sample was then analyzed for PrP^(Sc) and PrP^(C) protein contentby immunoblot. Two 100 μl aliquot of each sample was processed andelectrophoresed at 4° C. on an 8% polyacrylamide slab gel as describedin Example 2, i.e. under nondenaturing conditions. The gel is loadedwith two lanes of sample from the affected mouse and two lanes of samplefrom the control mouse, and preferable one lane of each is run on oneside of the gel, one lane of each on the other side of the gel, withwells containing no sample separating the two sides. The gel is run andtransferred to nylon as per Example 1.

Following transfer, the nylon is cut to separate the nylon into twoseparate blots, each containing a lane of affected and a lane of controlsample. Each nylon filter is placed in a heat-sealable plastic bag, and0.1 ml of blocking solution is added per square cm of filter. Theblocking solution contains 5% (w/v) nonfat dry milk (Carnation) and0.02% sodium azide in PBS. After 1 hour shaking at room temperature,antibody 3F4 is added to one blot in a 1:200 dilution, and antibody R1is added to the other blot in a 1:200 dilution, and the filter isincubated for 2-4 hours at 4° C. with gentle agitation on a platformshaker. Following incubation, the blocking solution and antibody areremoved, and each filter washed three times.

The secondary antibody is properly labeled to allow immunoblotdetection. The secondary antibody is added at a much more dilute levelthan the primary antibody, from 1:500 to 1:2000 dilution. Afterincubation, the filter is washed several times in PBS, each wash beingabout 10 minutes in length. The filter is then placed in a cassette witha piece of Xomat X-ray film (Kodak) at −70° C.

The level of PrP^(Sc) in the affected sample can be determined bycomparing the relative levels of signal of each antibody in the affectedversus the control mouse. This process can be a physical comparison todetermine the relative differences in signal level between the R1 andthe 3F4 antibody, or it may be a quantitative comparison. Comparisontechniques, both physical and quantitative, will be known to thoseskilled in the art. Quantitative comparison can be assessed using blotscanning techniques in which the levels are determined using computerprograms specially designed to assess comparative levels. One suchmethod is using a modified Excel spreadsheet program. Such programsallow for adjustments between samples based on variables such asbackground, time of hybridization, etc.

Levels of 3F4 staining should be consistent between the normal controlsamples of both the R1 and 3F4 antibody blots. The level of differencebetween R1 staining and 3F4 staining in the affected sample should allowthe quantification of PrP^(Sc) in the sample by subtracting the level ofPrP^(C) signal using the 3F4 antibody from the level of PrP^(Sc) andPrP^(C) signal from R1 antibody.

Such quantification may be adjusted based on antibody sensitivity,concentration, etc.

Example 5 Detection of βA4 in Human and Mouse Brain

A number of mouse models for Alzheimer's disease exhibit many of thehallmark protein changes associated with the human disease. Two examplesare: 1) mice with a modified human APP under the control of the PDGFpromoter (the “Athena-Lilly mouse”), the production and phenotype ofthese mice are described in U.S. Pat. No. 5,612,486, and 2) mice with amutant isoform of human APP under the control of the prion gene promoter(the “Hsiao mouse”) see Hsiao et al., Science 274:99-102 (1996). Thesemouse models for Alzheimer's disease display amyloid deposits and arecapable of producing all three major APP isoforms, and levels of APP andAP40/Aβ42 that progressively increase during the mouse's lifetime.

A 10% (w/v) brain homogenate from normal mice, affected Hsiao mice, andaffected Athena mice are prepared in TBS (25 mM Tris). After a low speedcentrifugation at 500×g for 15 min, the total protein in the supernatant(S1) is measured using a BCA Protein Assay (Pierce) and theconcentration is adjusted to 2.5 mg/ml with PBS.

A Dispase digestion is done at an enzyme to protein ratio of 1:25. Theprotein is digested with 100 μg/ml Dispase (Worthington) for 60 min at37° C. in the presence of 0.15% Zwittergent 3-12 (Calbiochem). Thedigestion is stopped by the addition of 50 mM EDTA. PAGE and transfer ofthe proteins is performed as in Example 1.

The nylon filter is placed in a heat-sealable plastic bag, and 0.1 ml ofblocking solution is added per square cm of filter. The blockingsolution contains 5% (w/v) nonfat dry milk (Carnation); 0.01% antifoamA; and 0.02% sodium azide in PBS. After 1 hour shaking at roomtemperature, a monoclonal antibody recognizing the β-amyloid protein,such as RDI-BAMYLOID (Research Daignostics) is added in a 1:200dilution, and the filter incubated for 2-4 hours at 4° C. with gentleagitation on a platform shaker. Following incubation, the blockingsolution and antibody are removed, and the filter washed three times.

The filter is incubated with an anti-mouse Ig secondary antibody inblocking solution for 1-2 hours at room temperature. The secondaryantibody is radiolabeled with to allow immunoblot detection. For theradiolabeled I¹²⁵ probe, approximately 10⁴ cm of the reagent is addedper square centimeter of filter. After incubation, the filter is washedseveral times in PBS, each wash being about 10 minutes in length. Thefilter is placed in a cassette with a piece of Xomat X-ray film (Kodak)at −70° C.

The resulting blot will have a band corresponding to βA4 in the sampleof the treated Athena Lilly or Hsiao mouse brain, both before and afterdigestion with Dispase. The sample of the normal mouse brain protein mayshow a low level band due to background with the APP protein. The lanewith the normal brain sample subjected to hydrolysis with Dispase shouldnot have a detectable band, since the Dispase will have hydrolysed thenormal protein in the sample.

This protocol may also be used with human brain or biopsy material, toidentify and diagnose individuals suspected of having Alzheimer'sdisease.

Example 6 Detection of TTR in Biopsy Material

A biopsy sample from the liver is taken from an individual thought to besuffering from familial amyloid polyneuropathy (FAP). Homogenates (10%(w/v)) of the liver sample and a normal liver control sample areprepared in TBS (25 mM Tris). After a low speed centrifugation at 500×gfor 15 min, the total protein in the supernatant (S1) is measured usinga BCA Protein Assay (Pierce) and the concentration is adjusted to 3.5mg/ml with PBS.

A Neprilysin digestion is done at an enzyme to protein ratio of 1:35.The protein is digested with 100 μg/ml Dispase for 60 min at 37° C. inthe presence of 0.2% Sarkosyl. The digestion is stopped by the additionof 50 mM EDTA. An aliquot of the proteins obtained in S1 both before andafter the neprilysin digestion is electrophoresed at 4° C. on an 8%polyacrylamide slab gel as described in Example 2, i.e. undernondenaturing conditions. The gel is run and proteins are transferred tonylon as in Example 1.

The nylon filter is placed in a heat-sealable plastic bag, and 0.1 ml ofblocking solution is added per square cm of filter. The blockingsolution contains 5% (w/v) nonfat dry milk (Carnation) and 0.02% sodiumazide in PBS. After 1 hour shaking at room temperature, a monoclonalantibody recognizing the amyloid conformation of TTR is added in a 1:100to 1:500 dilution, and the filter incubated for 2-4 hours at 4° C. withgentle agitation on a platform shaker. Following incubation, theblocking solution and antibody are removed, and the filter washed threetimes.

The secondary antibody is enzyme-conjugated with horseradish peroxidaseto allow immunoblot detection. The secondary antibody is added at a muchmore dilute level than the primary antibody, from 1:500 to 1:2000dilution. After incubation, the filter is washed several times in PBS,each wash being about 10 minutes in length. The filter is then placed ina cassette with a piece of Xomat X-ray film (Kodak) at −70° C.

The resulting blot will have a band corresponding to the amyloidconformation of TTR in an affected biopsy sample, but not in a biopsysample in which the amyloids are not present. Moreover, theconcentration of the TTR amyloid conformation may be a prognosticindicator as to the severity of the disease, and biopsies using multiplesamples may also be used to determine disease progression.

The instant invention is shown and described herein in what isconsidered to be the most practical, and preferred embodiments. It isrecognized, however, that departures may be made therefrom, which arewithin the scope of the invention, and that obvious modifications willoccur to one skilled in the art upon reading this disclosure.

1.-27. (canceled)
 28. A method of treating tissue, comprising the stepsof: removing a sample of brain tissue from an animal; homogenizing thesample to produce a homogenate; adding a dispase to the homogenate for aperiod of time and under conditions to allow for digestion of PrP^(C) inthe homogenate but leave PrP^(Sc) in the homogenate substantiallyundigested thereby providing a treated homogenate; analyzing the treatedsample to detect PrP^(Sc) in the treated sample.
 29. The method of claim27, wherein the animal is bovine.
 30. The method of claim 27, whereinthe dispase is a metalloendopeptidase dispase.
 31. The method of claim28, wherein the analyzing comprises contacting the treated sample with abinding partner that binds PrP^(Sc) in the treated sample.
 32. Themethod of claim 31, wherein the binding partner is an antibody.
 33. Themethod of claim 32, wherein the antibody is 3F-4.
 34. The method ofclaim 31, wherein the binding partner is attached to a detectable label.35. The method of claim 28, further comprising: inhibiting action of thedispase by addition of an inhibitor.
 36. The method of claim 35, whereinthe inhibitor is ethylene diaminetetracetic acid (EDTA).